Visual Progesterone Screening Immunoassay Test Kit Introduction Progesterone is a female sex hormone commonly referred to as the hormone of pregnancy for cow. The test is based on the expected changes in the production of hormone during the reproductive cycles and pregnancy. To use the milk progesterone test as a pregnancy test indicator, the milk must be collected between 21 days to 24 days after the cow was in estrus and inseminated. A cow with high progesterone cannot be in estrus and a cow with low progesterone cannot be pregnant. Reagents Materials Supplied with the kit: 1. 96 Anti-progesterone antibody coated microtitre wells 2. Reference standards: 1ml 2 ng/ml progesterone 3. 13 ml Enzyme Conjugate Reagent 4. 13 ml Solution A 5. 13 ml Solution B 6. 5 ml Stop solution (2N HCl) 7. droppers 8. test tubes for mixing solutions Materials needed ,but not supplied with the kit: 1. Paper towel or other absorbent paper Assay Procedure 1. Bring all components of the kit and milk samples to room temperature (25°C). 2. Break off the proper number of antibody coated wells (at least two for 2ng/ml progesterone standards plus two or more for each of the samples ) 3. In separate test tubes add two drop of each progesterone standard or milk sample, followed by three drops of Enzyme Conjugate Reagent to each tube. Mix well. 4. Transfer all of the solution to the antibody coated microtitre well. 5. Incubate for five minutes at room temperature. 6. After incubation, shake out the progesterone standard or milk sample/conjugate solution and rinse wells with water three times, making sure to sufficiently shake out the washings each time. Tap the wells on a paper towel to remove excess water. 7. add one drop of Solution A , followed by one drop of Solution B to each microtitre well. 8. Incubate at room temperature for 5 minutes. 9. Add one drop of Stopping solution (2N HCl) to each well. 10. Examine the color. Interpretation of Results 1. If the blue colour in the unknown samples is darker than the colour in the 2 ng/ml standard, then the sample contains less than 2 ng/ml of progesterone. 2. If the blue colour in the unknown samples is lighter than the colour in the 2ng/ml standard, then the sample contains more than 2 ng/ml of progesterone.